Already at a very early stage during evolution antibodies have been developed to protect the host organisms against invading molecules or organisms. Most likely one of the earliest forms of antibodies must have been developed in Agnatha. In these primitive fishes antibodies of the IgM type consisting of heavy and lights chains have been detected. Also in many other forms of life ranging from amphibians to mammals antibodies are characterized by the feature that they consist of two heavy and two light chains, although the heavy chains of the various classes of immunoglobulins are quite different. These heavy and light chains interact with each other by a number of different physical forces, but interactions between hydrophobic patches present on both the heavy and light chain are always important. The interaction between heavy and light chains exposes the complementarity determining regions (CDRs) of both chains in such a way that the immunoglobulin can bind the antigen optimally. Although individual heavy or light chains have also the capability to bind antigens (Ward et al., Nature 341 (1989) 544-546=ref. 5 of the above given draft publication) this binding is in general much less strong than that of combined heavy and light chains.
Heavy and light chains are composed of constant and variable domains. In the organisms producing immunoglobulins in their natural state the constant domains are very important for a number of functions, but for many applications of antibodies in industrial processes and products their variable domains are sufficient. Consequently many methods have been described to produce antibody fragments.
One of these methods is characterized by cleavage of the antibodies with proteolytic enzymes like papain and pepsin resulting in (a) antibody fragment comprising a light chain bound via an S—S bridge to part of a corresponding heavy chain formed by proteolytic cleavage of the heavy chain (Fab), or (b) a larger fragment of the antibody comprising two of these Fabs still connected to each other via an S—S bridge in enlargements of the heavy chain parts, indicated with F(ab)2, respectively (see patent applications EP-A-0125023 (GENENTECH/Cabilly et al., 1984) and WO-A-93/02198 (TECH. RES. CENT. FINLAND/Teeri et al., 1993) for definitions of these abbreviations). The disadvantage of the enzymatic route is that the production of whole antibodies is expensive and the enzymatic processing increases the costs of these fragments even more. The high costs of antibody fragments block the application of these fragments in processes and products outside the pharmaceutical industry.
Another method is based on, linkage on DNA level of the genes encoding (parts of) the heavy chain and the light chain. This linkage and the subsequent production of these chimeric immunoglobulins in microorganisms have been described (for Fab fragments see e.g. Better et al., Science 240 (1988) 1041-1043, for Fv fragments (combination of variable fragments of the heavy chain (VH) and light chain (VL) still connected to each other by non-covalent binding interactions) see e.g. Skerra et al., Science 240 (1988) 1938, and for single chain Fv fragments (ScFv; an Fv fragment in which the two variable fragments are linked to each other by a linker peptide) see e.g. Bird et al., Science 242 (1988) 423426. Provided that an appropriate signal sequence has been placed in front of the single chain VH and VL antibody fragment (ScFv), these products are translocated in E. coli into the periplasmic space and can be isolated and activated using quite elaborate and costly procedures. Moreover the application of antibody fragments produced by E. coli in consumer products requires extensive purification processes to remove pyrogenic factors originating from E. coli. For this and other reasons the production of ScFv in microorganisms that are normally used in the fermentation industry, like prokaryotes as Streptomyces or Bacillus (see e.g. Wu et al. Bio/Technology 11 (1993) 71) or yeasts belonging to the genera Saccharomyces (Teeri et al., 1993, supra), Kluyveromyces, Hansenula, or Pichia or moulds belonging to the genera Aspergillus or Trichoderma is preferred. However with a very few exceptions the production of ScFv antibodies using these systems proved to be impossible or quite poor. Although the exact reasons for the poor production are not well known, the use of linkers between the VH and VL chains not designed for secretion (Teeri et al., 1993, supra) may be a reason.
Another reason may be incorrect folding of ScFv. The frameworks and to a limited extend the CDRs of variable domains of light and heavy chains interact with each other. It has been described by Chothia et al. (J. Mol. Biol. 186 (1985) 651-663=ref. 13 of the above given draft publication) that this interaction involves amino acids at the following positions of the variable region of the heavy chain: 35, 37, 39, 44-45, 47, 100-103 and 105 (numbering according to Kabat et al., In “Sequences of Proteins of immunological Interest, Public Health Service, NIH, Washington D.C., 1983=ref 14 of the above given draft publication). Especially leucine at position 45 is strongly conserved and the whole a polar side chain of this amino acid seems to be involved in the interaction with the light chain. These strong interactions may fold the ScFv into a structure that can not be translocated in certain types of lower eukaryotes.
Thus the use of a linker in the production of ScFv for connecting a VH chain to a VL chain, might negatively influence either the translocation, or the folding of such ScFv or both.
Not prior-published European patent application 92402326.0 filed 21 Aug. 1992 (C. Casterman & R. Hamers) discloses the isolation of new animal-derived immunoglobulins devoid of light chains (also indicated as heavy chain immunoglobulins), which can especially originate from animals of the camelid family (Camelidae). This European patent specification, now publicly available as EP-A1-0 584 421, is incorporated herein by reference. These heavy chain immunoglobulins are characterized in that they comprise two heavy polypeptide chains sufficient for the formation of one or more complete antigen binding sites, whereby a complete antigen binding site means a site which will alone allow the recognition and complete binding of an antigen, which can be verified by any known method regarding the testing of the binding affinity. The European patient specification further discloses methods for isolating these heavy chain immunoglobulins from the serum of Camelidae and details of the chemical structure of these heavy chain immunoglobulins. It also indicates that these heavy chain immunoglobulins and derivatives thereof can be made by using recombinant DNA technology in both prokaryotes and eukaryotes. The present invention relates to a further development of the work disclosed in that prior-filed but not prior-published European specification.
Due to the absence of light chains in most of the immunoglobulins of Camelidae such linkers are not necessary, thereby avoiding the above-mentioned potential problems.
As described above in the draft publication for Nature, now publicly available as Nature 363 (3 Jun. 1993) 446-448, and in the not prior-published European patent application 92402326.0 (supra) it was surprisingly found that the majority of the protein A-binding immunoglobulins of Camelidae consists just of two heavy chains and that these heavy chains are quite different from common forms of heavy chains, as the CH1 domain is replaced by a long or short hinge (indicated for IgG2 and IgG3, respectively, in FIG. 4 of the above given draft publication for Nature). Moreover these heavy chains have a number of other features that make them remarkably different from the heavy chains of common immunoglobulins.
One of the most significant features is that they contain quite different amino acid residues at those positions involved in binding to the light chain, which amino acids are highly conserved in common immunoglobulins consisting of two heavy and two light chains (see Table 1 and SEQ. ID. NO: 13-31).
TABLE 1Comparison af amino acid sequences of variousimmunoglobulins Alignment of a number of V11 regions ofCamel heavy chain antibodies compared with those of mouse(M, top line) and human (H, second line). Frameworkfragments are indicated in capitals, CDR fragments in smallprint; see SEQ. ID. NO: 13-31 for sequences indicated by M,H, 1, 2, 3, 7, 9, 11, 13, 16, 17, 18, 19, 20, 21, 24, 25,27, 29, respectively.1                                                   50    mEVKLVESGGG LVQPGGSLRL SCATSGFTFS dfyme..WVR QPPGKRLEWI     hEVQLVESGGG LVQPGGSLRL SCAASGFTFS syams..WVR QAPGKGLEWV  cam1........GG SVQAGGSLRL SCAASGYSNC pltws..WYR QFPGTEREFV  cam2DVQLVASGGG SVQAGGSLRL SCTASGDSFS rfams..WFR QAPGKECELV  cam3........GG SVQTGGSLRL SCAVSGFSFS tscma..WFR QASGKQREGV  cam7........GG SVQGGGSLRL SCAISGYTYG sfcmg..WFR EGPGKEREGI  cam9........GG SVQAGGSLTL SCVYTNDTGT ...mg..WFR QAPGKECERV cam11........GG SVQAGGSLRL SCNVSGSPSS tyclg..WFR QAPGREREGV cam13........GG SVEAGGSLRL SCTASGYVSS ...ma..WFR QVPGQEREGV cam16........GG SAQAGGSLRL SCAAHGIPLN gyyia..WFR QAPGKGREGV cam17........GG SVQPGGSLTL SCTVSGATYS dysig..WIR QAPGKDREVV cam18........GG SVQAGGSLRL SCTGSGFPYS tfclg..WFR QAPGKEREGV cam19........GG SVQAGGSLRL SCAASDYTIT dycma..WFR QAPGKERELV cam20........GG SVQVGGSLRL SCVASTHTDS stcig..WFR QAPGKEREGV cam21........GG SVQVGGSLKL SCKISGGTPD rvpkslaWFR QAPEKEREGI cam24........GG SVQAGGSLRL SCNVSGSPSS tyclg..WFR QAPGKEREGV cam25........GG SVQTGGSLRL SCEISGLTFD dsdvg..WYR QAPGDECKLV cam27........GG SVQAGGSLRL SCASSSKYMP ctydmt.WYR QAPGKEREFV cam29.....eXXGG SVQAGGSLRL SCVASGFNFE tsrma..WYR QTPGNVCELV 51                                                 100    mA..asrnkan dytteysasv kgRFIVSRDT SQSILYLQMN ALRAEDTAIY     hS..xisxktd ggxtyyadsv kgRFTISRDN SKNTLYLQMN SLRAEDTAVY  cam1S..smd...p dgntkytysv kgRFTMSRGS TEYTVFLQMD NLKPEDTAMY  cam2S..siq...s ngrtteadsv qgRFTISRDN SRNTVYLQMN SLKPEDTAVY  cam3Aainsgggrt yyntyvaesv kgRFAISQDN AKTTVYLDMN NLTPEDTATY  cam7A..tiln..g gtntyyadsv kgRFTISQDS TLKTMYLLMN NLKPEDTGTY  cam9A..hit...p dgmtfidepv kgRFTISRDN AQKTLSLRMN SLRPEDTAVY cam11T..aint..d gsiiyaadsv kgRFTISQDT AKETVHLQMN NLQPEDTATY cam13A..fvqt..a dnsalygdsv kgRFTISHDN AKNTLYLQMR NLQPDDTGVY cam16A..ting..g rdvtyyadsv tgRFTISRDS PKNTVYLQMN SLKPEDTAIY cam17A..aant..g atskfyvdfv kgRFTISQDN AKNTVYLQMS FLKPEDTAIY cam18A..gins..a ggntyyadav kgRFTISQGN AKNTVFLQMD NLKPEDTAIY cam19A.aiqvvrsd trltdyadsv kgRFTISQGN TKNTVNLQMN SLTPEDTAIY cam20A..siyf..q dggtnyrdsv kgRFTISQLN AQNTVYLQMN SLKPEDSAMY cam21A..vlst..k dgktfyadsv kgRFTIFLDN DKTTFSLQLD RLNPEDTADY cam24T..aint..d gsviyaadsv kgRFTISQDT AKKTVYLQMN NLQPEDTATY cam25Sgilsdgtpy tksgdyaesv rgRVTISRDN AKNMIYLQMN DLKPEDTANY cam27S..sin...i dgkttyadsv kgRFTISQDS AKNTVYLQMN SLKPEDTAMY cam29S..siy...s dgktyyvdrm kgRFTISREN AKNTLYLQLS GLKPEDTAMY 101                                      139    mYCARdyygss .......y.. f.....dvWG AGTTVTVSS     hYCARXXXXXX xxxxxyyyyh x....fdyWG QGTLVTVSS  cam1YCKTalqpgg ycgygx.... ......clWG QGTQVTVSS  cam2YCGAvslmdr isqh...... ......gcRG QGTQVTVSL  cam3YCAAvpahlg pgaildlkky ......kyWG QGTQVTVSS  cam7YCAAelsggs celpllf... ......dyWG QGTQVTVSS  cam9YCAAdwkywt cgaqtggyf. ......gqWG QGAQVTVSS cam11YCAArltemg acdarwatla trtfaynyWG QGTQVTVSS cam13YCAAqkkdrt rwaeprew.. ......nnWG QGTQVTASS cam16FCAAgsrfss pvgstsrles .sdy..nyWG QGIQVTASS cam17YCAAadpsiy ysilxiey.. ......kyWG QGTQVTVSS cam18YCAAdspcym ptmpappird sfgw..ddFG QGTQVTVSS cam19SCAAtssfyw ycttapy... ......nvWG QGTQVTVSS cam20YCAIteiewy gcnlrttf.. ......trWG QGTQVTVSS cam21YCAAnqlagg wyldpnywls vgay..aiWG QGTHVTVSS cam24YCAArltemg acdarwatla trtfaynyWG RGTQVTVSS cam25YCAVdgwtrk eggiglpwsv qcedgynyWG QGTQVTVSS cam27YCXIdsypch ll........ ......dvWG QGTQVTVSS cam29YCAPveypia dmcs...... ......ryGD PGTQVTVSS
For example, according to Pessi et al. (1993) a subdomain portion of a VH region of common antibodies (containing both heavy chains and light chains) is sufficient to direct its folding, provided that a cognate VL moiety is present. Thus it might be expected from literature on the common antibodies that without VL chains proper folding of heavy chains cannot be achieved. A striking difference between the common antibodies and the Camelidae-derived heavy chain antibodies is, that the highly conserved apolar amino acid leucine (L) at place 45 present in common antibodies is replaced in most of the Camelidae-derived heavy chain antibodies by the charged amino acid arginine (R), thereby preventing binding of the variable region of the heavy chain to that of the light chains.
Another remarkable feature is that one of the CDRs of the heavy chains of this type of immunoglobulins from Camelidae. CDR3 is often much longer than the corresponding CDR3 of common heavy chains. Besides the two conserved cysteines forming a disulphide bridge in common VH fragments, the Camelidae VH fragments often contain two additional cysteine residues, one of which often is present in CDR3.
According to the present inventors these features indicate that CDR3 may play an important role in the binding of antigens by these heavy chain antibodies and can compensate for the absence of light chains (also containing CDRs) in binding of antigens by immunoglobulins in Camelidae.
Thus, as the heavy chains of Camelidae do not have special features for interacting with corresponding light chains (which are absent), these heavy chains are very different from common heavy chains of immunoglobulins and seem intrinsically more suitable for secretion by prokaryotic and lower eukaryotic cells.
The present inventors realized that these features make both intact heavy chain immunoglobulins of Camelidae and fragments thereof very attractive for their production by microorganisms. The same holds for derivatives thereof including functionalized fragment In this specification the term “functionalized fragment” is used for indicating an antibody or fragment thereof to which one or more functional groups, including enzymes and other binding polypeptides, are attached resulting in fusion products of such antibody fragment with another biofunctional molecule.